{"paper_id":"01c3a410-e6fd-4e65-bf5a-c4bd682db986","body_text":"An Effective Method for Bacterial Leaf Streak Disease Severity Estimation in Controlled and Field Environments in Small Grains | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article An Effective Method for Bacterial Leaf Streak Disease Severity Estimation in Controlled and Field Environments in Small Grains Muhammad Ahmad, Tapish Pawar, Shyam Solanki, Karl Glover, Gazala Ameen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8098615/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Wheat ( Triticum aestivum ) is one of the most economically important crops in the United States. However, over the past two years, wheat production has suffered up to a 40% reduction in final yield due to pathogen infections worldwide. A major emerging threat in the Great Plains and Canadian Prairies, including South Dakota, North Dakota, and Minnesota, is bacterial leaf streak (BLS)/black chaff disease caused by Xanthomonas translucens spp., which has led to substantial yield losses in the last decade. Absence of both effective chemical controls and competitive highly resistant varieties makes BLS disease management very difficult. A critical step missing in this process is the establishment of a reliable and reproducible infection protocol for resistance evaluation under both controlled and field conditions. Currently, no protocols are published, and the methods published as part of research manuscripts lack detailed procedures, equipment specifications, and have major drawbacks for applications limited to controlled environment and discrepancies in field disease ratings scales. Therefore, we are presenting here a robust and reproducible BLS disease infection protocol and, disease severity rating scale for estimation of BLS disease in both controlled and field conditions. Results After Three days of inoculation with X. translucens pv. undulosa ( Xtu ), wheat plants developed initial water-soaked symptoms at inoculation sites. Over seven days, symptoms progressed to chlorosis and necrosis, frequently covering entire leaves of highly susceptible genotypes, whereas limited to no symptoms on resistant genotypes. Disease severity was consistently scored on a 1–9 scale, enabling clear differentiation of resistant, moderately resistant, and susceptible genotypes. Pathogen re-isolation confirmed infection fidelity. Field validation at the booting stage produced comparable symptom progression on flag leaves, with severity scored at 7, 14, and 21 days post-inoculation. The same protocol was successfully adapted for Pantoea ananatis and Xanthomonas prunicola , demonstrating the adaptability of the method. The protocol was repeated across five independent trials and produced reproducible results in both controlled and field environments. Conclusion We describe a simple, reproducible, and cost-effective inoculation protocol for evaluating BLS severity in wheat. The method reliably distinguishes resistance responses across environments and can be extended to other bacterial pathogens affecting small grains. Its affordability, accessibility, and reproducibility make it a valuable tool for large-scale germplasm screening and resistance breeding. Key Features • A detailed and systemic infection protocol is devised for different cultivars of wheat. • Plants can screen at seedling and adult-plant stage. • No specific equipment required. • Using an inexpensive pipeline to ensure uniform symptoms. • This protocol is validated for other bacterial species that are reported to cause bacterial leaf streak symptoms on small grains ( Pantoea spp and Xanthomonas prunicola on small grains (wheat and Barley). Bacterial Leaf Streak Disease Wheat Xanthomonas translucence spp. Infection Assay Greenhouse Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background The global output of wheat and other cereals (barley, oats and corn) is hampered by bacterial plant disease (1). Bacterial leaf streak (BLS) disease of cereals (wheat, barley, rye, oat, and triticale) is caused by Xanthomonas translucens (Xt) . Typical symptoms of BLS include water-soaked, translucent streaks that progress into leaf necrotic lesions. In severe infections, the entire plant shows a blight appearance (2); thus this disease is also called bacterial blight. BLS on the grain heads leads to the darkening of kernels and is named black chaff. In the last decade, BLS incidence has dramatically increased in the Northern Great Plains (3, 4). In severe infections, the yield losses can reach up to 60 % on susceptible varieties (5). On average, in the US, about 14.5 million bushels of wheat are lost due to BLS and Black Chaff, which account for $78.5 million (Crop Protection Network, 2024). In 2018, and 2020 about 24 and 23 million bushels were lost to BLS, which accounted for $121 and $119 million lost in revenues (Crop Protection Network, 2024) ( Figure 2 and Table 1 ). Table 1 Estimated annual yield and economic losses in wheat due to bacterial leaf streak (BLS) in the United States from 2018 to 2024. Year % Loss Bushels Lost $(USD) Loss $(USD) Loss per Acre 2018 1.49% 23,585,844 $121,150,030 $3.10 2019 1.01% 18,251,357 $82,875,296 $2.01 2020 1.40% 23,047,060 $119,654,927 $2.98 2021* 0.03% 540,356 $3,491,449 $0.09 2022 0.49% 7,073,301 $64,979,800 $1.56 2023 0.43% 7,023,865 $51,561,573 $1.14 2024 0.46% 8,960,240 $49,751,075 $1.18 TOTALS 88,482,023 $493,464,149 AVERAGE 0.72% 12,640,289 $70,494,878 $1.72 *An odd year, where due to severe drought, data has not been collected on yield losses. Existent Infection Methodologies and Disease Severity Estimation Bacterial leaf streak disease management options are very limited. Several studies used chemicals like silicon (6) and copper containing compound (7) and more recently, 21 plant-protection products were evaluated to control BLS disease (8). However, none of the compounds showed consistent suppression of the BLS disease. Therefore, the most sustainable disease management solution is to develop a resistant variety against X. translucens . However, there is lack of detailed, robust, and reproducible protocol available to researchers for disease development. One method involved coating injured grains with the bacterial suspension, but it was considered labor intensive and unsatisfactory for pathogenicity testing (9). Another efficient approach is injecting a suspension into the leaf whorl of young plants (with 4-5 leaves) or into the boot of older plants using a hypodermic syringe. This technique has been validated at CIMMYT is often followed by incubation in a humid chamber (10). Other proposed methods include vacuum application (11). Using a specialized device to inject solution into thin plant leaves, which involves tongue seizing forceps, rubber stoppers and a hypodermic needle and syringe (12). Although leaf clippings and detached leaves have been used for pathogenicity testing using plants, inoculation and subsequent tests are generally considered effective (12). But all these methods are costly and time consuming. Another major issue in disease scoring lies in the inconsistency of rating scales used. The 1 to 5 scale from Acharya et al., (13) considers only water soaking and chlorosis, which does not represent the full range of symptoms observed under greenhouse and field conditions. In contrast, the 1 to 9 scale proposed in this protocol is more descriptive, employed for both seedling and adult-plant stages, and is consistent with the evaluation methods used for other important wheat diseases such as stripe rust, stem rust, spot blotch, and Soil-Borne Mosaic Virus (USDA Hard Winter Wheat Regional Nursery Programs). This broader and standardized approach provides a more reliable basis for disease estimation and would be widely accepted for application by both pathologists and breeders. Therefore, the protocol presented here is very timely, has the potential for wide adaptability and fills an essential knowledge gap for research needs against the bacterial leaf streak disease affecting small grains. Results and Discussion Bacterial Leaf Streak Disease Scoring in Controlled Environment Three days after inoculation with Xanthomonas translucens pv. undulosa ( Xtu ), the earliest symptoms were observed as water-soaked spots at the site of pathogen entry. By seven days post-inoculation, these lesions progressed into chlorotic areas and eventually developed necrotic tissue. In highly susceptible genotypes, the necrosis could expand to encompass the entire leaf. Symptom development was reliably captured using a 1–9 visual scoring scale (Fig. 3 ), which allowed differentiation of resistant and susceptible responses. Diseased leaves were collected and brought to the laboratory, where the presence of Xtu was confirmed through pathogen isolation, validating that the observed symptoms were indeed caused by the inoculated bacterium. This assay was repeated five times, and the results were highly consistent, demonstrating the robustness and reproducibility of the inoculation method. Bacterial Leaf Streak Disease Scoring in Field Environment Field evaluation focused on the flag leaf, which is critical for grain filling and particularly sensitive to pathogen-induced damage. Disease severity was quantified using a standardized 1–9 scale (Fig. 4 ), where scores 1 indicated resistance and scores 9 indicated susceptibility. Plants with scores of 1–3 exhibited either no visible symptoms or only faint streaks restricted to a small portion of the leaf, indicative of resistant reactions. Moderate susceptibility (scores 4–6) was characterized by distinct water-soaked streaks affecting less than half of the leaf area. Highly susceptible plants (scores 7–9) showed extensive streaking and necrosis, often coalescing into large, blighted areas that covered most or all the flag leaf. The use of this standardized scoring system enabled reliable comparison of disease responses across multiple genotypes and field replicates, providing a clear framework for identifying resistant and susceptible wheat lines. Applications of the Protocol in Multiple Field Trials of Wheat We conducted multiple field trials in hard red spring and winter wheat and durum wheat and barley to evaluate disease severity following artificial inoculation with Xtu , Xanthomonas prunicola, Pantoea ananatis , and Pantoea agglomerans . In one of the experiments conducted, we used the same set of wheat genotypes and inoculated across environments to assess the consistency of this protocol, symptoms and host response. In wheat, inoculation with Xtu produced characteristic bacterial leaf streak (BLS) symptoms, with water-soaked lesions that later became necrotic and streak-like, particularly under favorable environmental conditions. In the inoculated field plots, clear differences between resistant and susceptible wheat genotypes were observed (Fig. 5A). The resistant and susceptible lines grown side by side demonstrated the accuracy and consistency of the inoculation protocol. Susceptible lines exhibited rapid disease progression, leading to coalesced streaks and premature leaf senescence. In severe cases, entire leaves dried early, resulting in substantial canopy damage and potential yield reduction (Fig. 5B). Moreover, blight-like symptoms extended beyond the foliage, as black chaff appeared on wheat spikes, marked by dark, water-soaked, and necrotic lesions on glumes and awns (Fig. 5C). These symptoms were most pronounced in genotypes displaying high foliar susceptibility to Xtu , suggesting systemic infection and pathogen movement within the plant. In contrast, inoculation with X . prunicola had more symptoms compared to Xtu and P. ananatis which showed the complexity of this disease, X. prunicola produced more necrotic spots than translucence streak, while P. ananatis symptoms were generally less severe across replicates. While chlorosis and occasional necrotic flecks were observed on P. ananatis inoculated wheat genotypes, the extent of coalescing necrotic leaves did not approach to a score of 9. Thus, P. ananatis strain tested, failed to induce clear streak symptoms under field conditions, indicating either weak pathogenicity or a role more consistent with opportunistic colonization. Collectively, these results confirms that Xtu remains highly virulent for both wheat and barley in the US, capable of causing both foliar BLS and black chaff on spikes and with new species of Xanthomonas detected in the US, the BLS disease is complex and emerging in both South and North American continents ( 14 , 15 ). Black Chaff Symptoms caused by Bacterial Leaf Streak Disease on spikes In addition to foliar BLS symptoms, clear manifestations of black chaff were observed on wheat spikes following inoculation with Xtu . Symptoms included dark brown to black streaks and blotches on the glumes and awns, with lesions often extending longitudinally along spikelet (Fig. 6 A). In severely affected spikes, symptoms intensified, with coalesced streaks and widespread necrosis on glumes and awn, giving a pronounced blackened appearance (Fig. 6 B). The severity of black chaff was positively correlated with foliar susceptibility, as highly susceptible lines exhibiting intense BLS also showed more severe black chaff on spikes. Inoculations with X. prunicola , Pantoea ananatis , and Pantoea agglomerans did not reproduce typical black chaff symptoms, producing either mild discoloration or no visible spike damage. These findings confirm that black chaff is a diagnostic symptom of X tu infection in wheat and highlight its importance as a phenotypic marker for assessing susceptibility and yield impact under field conditions. Application of Developed Methodology in Controlled Environment The inoculation methodology successfully induced consistent BLS symptoms under greenhouse conditions. Water-soaked lesions appeared within three days of inoculation, progressing to chlorosis and necrosis within 7–10 days. Disease severity, scored on a 1–9 scale, showed a wide range of responses across genotypes. Violin plots (Fig. 7 A, B) demonstrated clear separation between resistant, moderately resistant, and susceptible groups. Resistant genotypes were concentrated at lower scores ( 1 – 3 ), while susceptible lines showed higher distributions ( 7 – 9 ). The statistical analysis confirmed these phenotypic differences. In Experiment 1, variation due to treatment was marginally significant (F = 2.88, p = 0.0571), indicating some experimental influence ( Supplementary Table 3 ). However, in Experiment 2, highly significant differences were observed among genotypes (F = 7.30, p < 2e − 16), with genotype effects explaining most of the variance compared to residual error ( Supplementary Table 4 ). These findings demonstrate that while environmental or experimental variation can influence disease expression, the methodology consistently discriminates among resistant and susceptible genotypes under greenhouse conditions. Field Experiments Field validation under natural infection pressure supported the robustness of the developed method. Both flag and bottom leaves were evaluated, with bottom leaves generally exhibiting higher severity scores, reflecting natural disease progression (Fig. 8 A, B). The bar chart (Fig. 8 A) showed that most genotypes clustered around moderate severity classes ( 4 – 6 ), while violin plots (Fig. 8 B) revealed clear distinctions between resistant and susceptible lines. ANOVA further confirmed significant variation among experimental factors. In the first field trial, strong effects of trial (F = 67.98, p < 2e − 16) and day (F = 1296.0, p < 2e − 16) were detected, with a smaller but significant trial × day interaction (F = 2.74, p = 0.0118) ( Supplementary Table 5 ). These results indicate that disease development was influenced by both environment and scoring time, but the magnitude of genotypic effects remained much stronger. In Experiment II, highly significant differences among wheat lines were observed (F = 2.13, p < 2e − 16), confirming that genetic variability was the primary determinant of BLS severity ( Supplementary Table 6 ). Together, these results demonstrate that the methodology is reproducible in both greenhouse and field conditions, allowing reliable identification of resistant, moderately resistant, and susceptible genotypes. The combined evidence from distribution patterns and ANOVA strongly supports its use in large-scale resistance screening programs. Discussion The lack of a reliable and reproducible inoculation protocol has long been a major constraint in advancing research on bacterial leaf streak (BLS) and black chaff of small grains. The inability to consistently reproduce infections under controlled conditions has limited progress in elucidating host-pathogen interactions, identifying resistance sources, and accelerating breeding for resistant cultivars. Although BLS has been recognized in the United States for several decades, it has emerged as a serious threat in the Northern Great Plains in recent years ( 16 ). Despite increasing importance, reproducible inoculation methods remain challenging, with most previous approaches such as needleless syringe or vacuum infiltration and leaf clipping, requiring specialized facilities and operator skill while yielding variable amounts of bacteria inoculum, disease severity outcomes, labor intensive and time-consuming ( 3 , 7 ). The disease rating scales currently published consider only leaf water-soaking and chlorosis and do not account for necrosis symptoms. Such inconsistency in both inoculation protocols, disease severity and rating scales has hindered the fulfillment of Koch’s postulates and slowed systematic screening for resistance. Earlier studies demonstrated that inoculation method, bacterial concentration, and plant growth stage significantly influence symptom development and disease severity. Syringe or vacuum infiltration methods force bacterial entry into leaf apoplast but often cause mechanical injury and fail to represent natural infection capabilities of bacterial strains. Conversely, spray inoculations more closely mimic field conditions but often result in uneven droplet distribution and poor bacterial adherence to the leaf surface, particularly when surfactants are absent. Environmental parameters such as humidity, temperature, and dew duration after inoculation also critically affect lesion formation [1, 3]. In several reports, bacterial suspensions below 10⁵ CFU mL⁻¹ failed to produce consistent symptoms, whereas higher concentrations (≥ 10⁶ CFU mL⁻¹) induced typical streaking in susceptible cultivars ( 13 ). These findings underscore the importance of standardizing inoculum preparation, delivery, and environmental conditions for reliable infection establishment. The air-pressure spray inoculation method described in this study addresses all of these limitations by providing a scalable and reproducible approach suitable for both controlled and field environments. Incorporating a non-ionic surfactant into the bacterial suspension ensures uniform wetting and adhesion of droplets to leaf surfaces, facilitating bacterial penetration through stomata and cuticular openings. The controlled air pressure allows consistent droplet size and uniform distribution of inoculum across leaves and spikes, producing reproducible symptom development across genotypes. In susceptible cultivars, water-soaked lesions typically appeared three days post-inoculation and progressed to chlorosis and necrosis within seven days, whereas resistant lines exhibited minute, restricted lesion expansion. This consistency supports the method’s utility for screening germplasm and differentiating resistance levels. Notably, when applied at booting, the protocol also successfully reproduced black chaff symptoms on spikes, providing a comprehensive assessment of both foliar and reproductive stage susceptibility, a feature absent from many previous studies ( 7 , 10 ). The broader applicability of this inoculation system was demonstrated by its successful adaptation to other bacterial pathogens, including X . prunicola and P. ananatis. While Xtu remains the primary causal agent of severe BLS in U.S. wheat, emerging reports of X. prunicola and Pantoea species highlight a complex disease etiology ( 3 ). The adaptability of the current protocol to multiple pathogens emphasizes its flexibility and potential use for comparative pathogenicity testing and cross-host inoculation studies in small grains. A further contribution of this work is the standardization of disease scoring. Previous studies often employed a 1–5 scale that captured early water soaking and chlorosis but failed to represent advanced necrotic or spike symptoms ( 17 ). The 1–9 scale implemented here provides higher resolution and aligns BLS assessment with scoring systems used for other major wheat diseases such as stripe rust, stem rust, soil-borne mosaic virus and spot blotch. This harmonization enables comparative evaluation of resistance across diseases and improves the integration of phenotypic data into practical applied pathology and breeding. Despite these advantages, the method requires careful management of inoculum quality, environmental humidity, and plant growth stage to ensure optimal results. Field disease severity was greatest when inoculation occurred at booting, suggesting that physiological and anatomical changes during spike emergence influence bacterial colonization. This observation aligns with previous reports that host developmental stage affects infection success and symptom expression ( 10 , 17 ). Further refinement is needed to define the latent epiphytotic period, quantify bacterial proliferation within tissues during pathogenic stage, and assess the influence of environmental variability across locations. Overall, the air-pressure spray inoculation method represents a significant methodological advance for BLS and black chaff research. Its reproducibility, scalability, and adaptability make it suitable for both fundamental studies on Xanthomonas translucens pathogenesis and large-scale resistance screening in pathology and breeding research. Moreover, the approach can be extended to related cereal hosts and emerging bacterial pathogens, positioning it as a versatile and standardized tool for the wider plant research community. Conclusions This study establishes a reliable, reproducible, and cost-effective assay for bacterial leaf streak (BLS) evaluation in wheat. The protocol consistently reproduced characteristic symptoms under both greenhouse and field conditions, enabling clear differentiation between resistant and susceptible genotypes using a standardized 1 to 9 scale. Multiple repetitions confirmed the robustness, accuracy and adaptability of this method, not only for Xtu but also for related pathogens such as X. prunicola and P. ananatis . Unlike earlier approaches that were not accurate, labor-intensive, or dependent on specialized equipment, this assay is accurate, simple, scalable, and efficient for large germplasm screening. By integrating greenhouse and field evaluations, the protocol provides a powerful tool to accelerate discovering key components of the pathogen effectors, host resistance genes, plant-bacterial interactions, yield loss estimation, and breeding efforts aimed at developing BLS-resistant varieties. Importantly, it also demonstrates potential utility for other cereals and bacterial diseases, broadening its impact on cereal pathology and crop improvement. Key Implications The development of this simple yet robust assay directly benefits plant biologists, breeders, pathologists, and agronomists by offering an efficient way to screen wheat germplasm for resistance against BLS. Its flexibility across pathogens and cereal crops provides a unified framework for studying streak-like bacterial diseases, which are increasingly threatening cereal production worldwide. Most importantly, this protocol bridges-controlled environment and field evaluations, ensuring reliable disease phenotyping that can accelerate research. Ultimately, the adoption of this method will contribute to accelerated breeding for BLS resistance, sustainable wheat production, reduced yield losses, and enhanced global food security. Material and Methods Xtu Isolation and Characterization The diseased samples were collected from the Volga SDSU wheat field station and the Dakota Lakes Research Farm on July 9th, 2023. The diseased samples were cut into small pieces and surface sterilized with 10% bleach and 70% ethanol, followed by ddH2O washing ( 7 ). The leaf was crushed into ddH 2 O and with the help of a sterile loop, streaked on nutrient agar (NA) ( Supplementary Table 1 ) and kept in an incubator for 2 days at 28°C. The single yellowish colony streaked separately on NA plates for pure culture. The DNAs were extracted from all single colonies by using the protocol of ( 18 ) for Multiplex primer amplification specific to Xanthomonas translucence spp. and 16S rRNA gene sequencing. Culture Preparation and Greenhouse Inoculation After the confirmation of Xtu AL-1029 (Accession No. PQ524066) ( 19 ) the secondary culture was grown in NA media supplemented with 10% sucrose. Bacterial cells were then suspended in 1X phosphate saline buffer (PBS) (pH 7.4) to prepare an inoculum. To achieve an optical density (OD 600 ) of 0.4, or around 1×10 8 cfu/ml, the cell concentration was adjusted. Four drops of Tween-20 (polyoxyethylene sorbitan monolaurate, Sigma-Aldrich) were added as a surfactant to the bacterial solution before inoculation. The plants were grown in 9-inch cones with three replications, and each replication had two plants, total six plants were inoculated after 15 days of germination. The bacterial solution was equally sprayed over the plants till runoff for the real inoculation procedure. A spray cannon connected to an air pump with around 20psi of pressure. Following this, the infected plants were kept at room temperature and subjected to a 12-hour photo period in misting chambers for two days. The plants were moved and placed on flow trays within the greenhouse room at 80% humidity for seven days. The assessment of disease development was scored by a scale from 1 to 9 (1 for highly Resistant and 9 for highly susceptible), with an estimation made of the proportion of the plant area affected by the disease. Field Inoculation Assay The same protocol was used for inoculating the field trial. Inoculation was carried out at the wheat booting stage using an air-pressure sprayer, without the addition of carborundum powder. Disease severity was assessed at 7th, 14th, and 21st days post-inoculation ( Supplementary Fig. 1 ), scoring the flag leaf on a 1–9 BLS disease scale. The same method was applied to screen Xanthomonas prunicola ( 15 ) and Pantoea ananatis ( 20 ) in the field. Since BLS is a disease complex, this protocol can be reliably used to evaluate all bacterial pathogens that produce streak-like symptoms on small grains plants. In summer 2024 and 2025, more than 1000 row plots of winter, spring and durum wheat and winter barley were inoculated with AL-1029 Xtu strain by following the same protocol. There was one repeated experiment in 2024 and 2025 where the same set of genotypes was inoculated with Xtu , P. ananatis and X. prunicola by following the same protocol. Development of the new Methodology Here, we provide a detailed, step-by-step procedure for small-scale, repeatable BLS infection testing in wheat. The procedure was created to treat wheat that had been infected with Xanthomonas translucence spp. It comprises a pre-propagation and simple harvesting process to generate a large enough quantity for an infection trial. The technique may be quickly established because of the thorough process of knowledge and ease of handling. The necessary tools are easily accessible and reasonably priced. In addition, we show that a particular resistance phenotype may be evaluated by combining our infection strategy with macroscopic and microscopic assessment techniques. Finally, the procedure is easily adaptable to various host plants and bacterial and fungal diseases. Diseased wheat leaves showing typical streak and necrosis symptoms were collected from the field (Fig. 9 A). Collected leaves were cut into small pieces, surface-sterilized to eliminate epiphytic microbes, and macerated in 500 µL sterile water. The suspension was streaked onto nutrient agar (NA) plates and incubated at 28°C for 2 days (Fig. 9 B). Single colonies were sub-streaked onto fresh NA plates to establish pure cultures; a process repeated three times to ensure purity. The identity of X. translucens pv. undulosa ( Xtu ) was confirmed to be using published multiplex primers ( 2 ) and agarose gel electrophoresis (Fig. 9 C). Pure Xtu cultures were streaked onto NA plates supplemented with 10% sucrose and incubated for 5–7 days to obtain maximum bacterial growth prior to inoculation (Fig. 9 D). A 1× phosphate-buffer saline (PBS) solution ( Supplementary Table 2 ) was prepared and autoclaved. Bacterial colonies were scraped from the NA plates, resuspended in 1X PBS, and adjusted to an optical density (OD 600 ) of ~ 0.4, corresponding to 1 × 10 8 cfu/mL. Four drops of Tween-20 were added per 100 mL of inoculum (Fig. 9 E). Wheat plants were grown in 9-inch cones, each containing two seeds, with three replications (total: 6 plants per treatment). Plants were maintained under controlled conditions (23–25°C, 14 h light/10 h dark photoperiod) for 15 days, with adequate watering and fertilizer (Fig. 9 F). Inoculation was performed 15 days after germination. The prepared inoculum was applied evenly to the plants using an air sprayer set at 25 psi (Fig. 9 G). Inoculated plants were incubated in a humidity chamber at 25°C with 100% relative humidity and a 14 h light/10 h dark cycle for 48 h (Fig. 9 H). Following incubation, plants were transferred to a transparent tent maintained at ~ 80% relative humidity with the aid of a humidifier, where they remained for 7–10 days to allow disease development (Fig. 9 I). Disease severity was assessed 7 days post-inoculation using a 1–9 rating scale (Fig. 4 ) initial symptoms were gummy like exudates produced on leaf surface (Fig. 9 J, K), these exudates progressed into translucence streak (Fig. 9 L) which later turned necrotic lesion (Fig. 9 M). Abbreviations BLS Bacterial leaf streak Xtu Xanthomonas translucence pathovar undulosa Xp Xanthomonas prunicola PBS Phosphate buffer saline cfu Colony forming unites AL1029 Ameen Lab strain 1029 DPI Day post Inoculation NA Nutrient agar SDSU South Dakota State University Declarations Acknowledgements: We thank Mr. Jack Ingemansen, Steve Stein, Cody Hall, Chris Nelson, Julie Thomas and Dr. Dalitso Yabwalo for help with field planting, preparations and management of research plots. Authors Information: Authors and Affiliations Department of Agronomy, Horticulture & Plant Science, South Dakota State University, Brookings, South Dakota, 57007, U.S.A. Muhammad Ahmad, Tapish Pawar, Shyam Solanki, Karl Glover and Gazala Ameen Contributions MA planned and designed the experiments, conducted research, analyzed the data, prepared figures and wrote the manuscript. GA, MA and KG develop the protocol. MA and TP conducted the field trials and collected data. GA and KG gathered funding for the projects. SS, TP, KG and GA reviewed the manuscript, edited and provided the ideas for improvement of the technique and manuscript. Corresponding author Correspondence to Gazala Ameen Funding This study was carried out with the support of the Agricultural Experimentation Funding SD00H754, SD00H717, and South Dakota Wheat Commission funding SA2400069 and SA2500056. Ethics Declarations Ethics approval and consent to participate Not applicable Consent for Publication All the authors have reviewed and edited this manuscript and agreed to submit it to the Plant Method Journal for publication. 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Maps on the left show economic losses (USD), while maps on the right display losses per acre ($/ac). Loss intensity is represented by color gradients, with darker shades indicating higher losses (Crop Protection Network, 2024).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/b563d44b405301c23fdbc3a7.png\"},{\"id\":96661026,\"identity\":\"75ea429c-4284-4f76-883e-a5f38b3d334c\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 18:26:30\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":680590,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDisease severity ranging from 1 to 9 of third leaf of wheat plant after 15 days of inoculation. 1 indicate the resistant and 9 indicate the susceptible against \\u003cem\\u003eXanthomonas translucens \\u003c/em\\u003epathovar\\u003cem\\u003e undulosa.\\u003c/em\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/57b0935a4ea2c19066d9970f.png\"},{\"id\":96661027,\"identity\":\"2189bc4a-58c3-4faa-8561-966b7a05171c\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 18:26:30\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":912214,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBacterial Leaf Streak disease severity ranging from 1 to 9 of flag leaf of wheat plant after 21\\u003csup\\u003est\\u003c/sup\\u003e days of inoculation. 1 indicate the resistant and 9 indicate the susceptible against \\u003cem\\u003eXanthomonas translucens \\u003c/em\\u003epv\\u003cem\\u003e undulosa.\\u003c/em\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/5d258e417e5ddfeb7b238d43.png\"},{\"id\":96661032,\"identity\":\"52621476-400c-441d-a2cc-38bd9203b610\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 18:26:30\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":726535,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInoculated wheat field plot showing the Bacterial Leaf Streak symptoms. (A) Comparison of resistant and susceptible wheat genotypes showing the accuracy of the protocol. (B) Moderately resistant wheat genotype (C) Blight-like appearance on susceptible wheat genotype.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/9e67355ffac7c53af0e8a3ac.png\"},{\"id\":96709521,\"identity\":\"b34fe49a-adfd-4a54-b1b7-7aa9e5abc556\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:09:11\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":602973,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBlack chaff symptoms on wheat spikes following inoculation with \\u003cem\\u003eXanthomonas translucens\\u003c/em\\u003e pv. \\u003cem\\u003eundulosa\\u003c/em\\u003e. (A)Early-stage black chaff showing streaking and blotching on glumes and awns. (B) Severe black chaff with extensive dark streaking and coalesced necrosis on spike tissues.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/3706f9af28f4b5b8f0716dec.png\"},{\"id\":96661028,\"identity\":\"8de77287-3a92-482e-b5ff-0c2de86037d8\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 18:26:30\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":126088,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDistribution of bacterial leaf streak (BLS) disease severity scores in controlled environment represented by violin plots with embedded boxplots. The violin shape indicates the density of observations across the 1–9 rating scale, while individual data points are overlaid to show variation within groups. N shows the number of genotypes used for testing.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/127e1463f3749c55eb965a3b.png\"},{\"id\":96661029,\"identity\":\"3c7ab884-f029-4d57-94a7-5822fbe30960\",\"added_by\":\"auto\",\"created_at\":\"2025-11-24 18:26:30\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":115314,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eField evaluation of bacterial leaf streak (BLS) severity in wheat. (A) Violin plots and frequency distribution of disease scores (1–9 scale) recorded on flag and bottom leaves across genotypes, showing differential levels of resistance and susceptibility in first experiment. (B) Boxplots and line graph comparing disease scores across independent trials, illustrating consistency and variation in disease responses for 2\\u003csup\\u003end\\u003c/sup\\u003e experiment. N shows the number of genotypes used for the BLS disease screening.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/14b60c76807661f554cbd6be.png\"},{\"id\":96709828,\"identity\":\"635dd909-9ba8-4ece-a2af-446983a3a76e\",\"added_by\":\"auto\",\"created_at\":\"2025-11-25 10:09:43\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":804332,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStepwise workflow for bacterial leaf streak (BLS) inoculation and infection assay in wheat.\\u003c/p\\u003e\\n\\u003cp\\u003e(A)Wheat leaves showing typical BLS symptoms with chlorotic and necrotic streaks. (B) Initial bacterial colonies from field-infected leaf tissue plated on nutrient agar. (C–D)Purified single colonies of \\u003cem\\u003eXtu\\u003c/em\\u003e obtained by streak plating. (E) Liquid culture of \\u003cem\\u003eXtu\\u003c/em\\u003e prepared in PBS buffer for inoculum. (F) Healthy wheat seedlings grown under controlled conditions prior to inoculation. (G)Inoculation of wheat plants using foliar spray with bacterial suspension. (H–I) High-humidity chambers used to maintain favourable conditions for infection and disease development. (J–L) Early symptom development (red arrows) showing water-soaked lesions and streak formation on leaves. (M) Representative wheat leaves showing advanced BLS symptoms used for disease scoring.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/8c83b8b48e1d37cf644a513b.png\"},{\"id\":98421927,\"identity\":\"8ce3d150-9d7d-40d1-8fbd-ec82bb4723da\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:29:56\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":6429913,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8098615/v1/c0b98c85-3173-454f-9b14-151dd1ba6284.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"An Effective Method for Bacterial Leaf Streak Disease Severity Estimation in Controlled and Field Environments in Small Grains\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eThe global output of wheat and other cereals (barley, oats and corn) is hampered by bacterial plant disease (1). Bacterial leaf streak (BLS) disease of cereals (wheat, barley, rye, oat, and triticale) is caused by \\u003cem\\u003eXanthomonas translucens (Xt)\\u003c/em\\u003e. Typical symptoms of BLS include water-soaked, translucent streaks that progress into leaf necrotic lesions. In severe infections, the entire plant shows a blight appearance (2); thus this disease is also called bacterial blight. BLS on the grain heads leads to the darkening of kernels and is named black chaff. In the last decade, BLS incidence has dramatically increased in the Northern Great Plains (3, 4). In severe infections, the yield losses can reach up to 60 % on susceptible varieties (5). On average, in the US, about 14.5 million bushels of wheat are lost due to BLS and Black Chaff, which account for $78.5 million (Crop Protection Network, 2024). In 2018, and 2020 about 24 and 23 million bushels were lost to BLS, which accounted for $121 and $119 million lost in revenues (Crop Protection Network, 2024) (\\u003cstrong\\u003eFigure 2 and Table 1\\u003c/strong\\u003e).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTable 1\\u0026nbsp;Estimated annual yield and economic losses in wheat due to bacterial leaf streak (BLS) in the United States from 2018 to 2024.\\u003c/p\\u003e\\n \\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"605\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eYear\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e% Loss\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eBushels Lost\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e$(USD) Loss\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e$(USD) Loss per Acre\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2018\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e1.49%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e23,585,844\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$121,150,030\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$3.10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2019\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e1.01%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e18,251,357\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$82,875,296\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$2.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2020\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e1.40%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e23,047,060\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$119,654,927\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$2.98\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2021*\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e0.03%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e540,356\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$3,491,449\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$0.09\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2022\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e0.49%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e7,073,301\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$64,979,800\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$1.56\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2023\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e0.43%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e7,023,865\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$51,561,573\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$1.14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e2024\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e0.46%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e8,960,240\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$49,751,075\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$1.18\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTOTALS\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e88,482,023\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$493,464,149\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAVERAGE\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 17.9868%;\\\"\\u003e\\n \\u003cp\\u003e0.72%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e12,640,289\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 18.4818%;\\\"\\u003e\\n \\u003cp\\u003e$70,494,878\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 27.0627%;\\\"\\u003e\\n \\u003cp\\u003e$1.72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003e*An odd year, where due to severe drought, data has not been collected on yield losses.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eExistent Infection Methodologies and Disease Severity Estimation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBacterial leaf streak disease management options are very limited. Several studies used chemicals like silicon (6) and copper containing compound (7) and more recently, 21 plant-protection products were evaluated to control BLS disease (8). However, none of the compounds showed consistent suppression of the BLS disease. Therefore, the most sustainable disease management solution is to develop a resistant variety against\\u003cem\\u003e\\u0026nbsp;X. translucens\\u003c/em\\u003e. However, there is lack of detailed, robust, and reproducible protocol available to researchers for disease development. One method involved coating injured grains with the bacterial suspension, but it was considered labor intensive and unsatisfactory for pathogenicity testing (9). Another efficient approach is injecting a suspension into the leaf whorl of young plants (with 4-5 leaves) or into the boot of older plants using a hypodermic syringe. This technique has been validated at CIMMYT is often followed by incubation in a humid chamber (10). Other proposed methods include vacuum application (11). Using a specialized device to inject solution into thin plant leaves, which involves tongue seizing forceps, rubber stoppers and a hypodermic needle and syringe (12). Although leaf clippings and detached leaves have been used for pathogenicity testing using plants, inoculation and subsequent tests are generally considered effective (12). But all these methods are costly and time consuming. Another major issue in disease scoring lies in the inconsistency of rating scales used. The 1 to 5 scale from Acharya et al., (13) considers only water soaking and chlorosis, which does not represent the full range of symptoms observed under greenhouse and field conditions. In contrast, the 1 to 9 scale proposed in this protocol is more descriptive, employed for both seedling and adult-plant stages, and is consistent with the evaluation methods used for other important wheat diseases such as stripe rust, stem rust, spot blotch, and Soil-Borne Mosaic Virus (USDA Hard Winter Wheat Regional Nursery Programs). This broader and standardized approach provides a more reliable basis for disease estimation and would be widely accepted for application by both pathologists and breeders. Therefore, the protocol presented here is very timely, has the potential for wide adaptability and fills an essential knowledge gap for research needs against the bacterial leaf streak disease affecting small grains.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003eBacterial Leaf Streak Disease Scoring in Controlled Environment\\u003c/h2\\u003e\\n \\u003cp\\u003eThree days after inoculation with \\u003cem\\u003eXanthomonas translucens\\u003c/em\\u003e pv. \\u003cem\\u003eundulosa\\u003c/em\\u003e (\\u003cem\\u003eXtu\\u003c/em\\u003e), the earliest symptoms were observed as water-soaked spots at the site of pathogen entry. By seven days post-inoculation, these lesions progressed into chlorotic areas and eventually developed necrotic tissue. In highly susceptible genotypes, the necrosis could expand to encompass the entire leaf. Symptom development was reliably captured using a 1\\u0026ndash;9 visual scoring scale (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e), which allowed differentiation of resistant and susceptible responses. Diseased leaves were collected and brought to the laboratory, where the presence of \\u003cem\\u003eXtu\\u003c/em\\u003e was confirmed through pathogen isolation, validating that the observed symptoms were indeed caused by the inoculated bacterium. This assay was repeated five times, and the results were highly consistent, demonstrating the robustness and reproducibility of the inoculation method.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eBacterial Leaf Streak Disease Scoring in Field Environment\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eField evaluation focused on the flag leaf, which is critical for grain filling and particularly sensitive to pathogen-induced damage. Disease severity was quantified using a standardized 1\\u0026ndash;9 scale (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), where scores 1 indicated resistance and scores 9 indicated susceptibility. Plants with scores of 1\\u0026ndash;3 exhibited either no visible symptoms or only faint streaks restricted to a small portion of the leaf, indicative of resistant reactions. Moderate susceptibility (scores 4\\u0026ndash;6) was characterized by distinct water-soaked streaks affecting less than half of the leaf area. Highly susceptible plants (scores 7\\u0026ndash;9) showed extensive streaking and necrosis, often coalescing into large, blighted areas that covered most or all the flag leaf. The use of this standardized scoring system enabled reliable comparison of disease responses across multiple genotypes and field replicates, providing a clear framework for identifying resistant and susceptible wheat lines.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eApplications of the Protocol in Multiple Field Trials of Wheat\\u003c/h3\\u003e\\n\\u003cp\\u003eWe conducted multiple field trials in hard red spring and winter wheat and durum wheat and barley to evaluate disease severity following artificial inoculation with \\u003cem\\u003eXtu\\u003c/em\\u003e, \\u003cem\\u003eXanthomonas prunicola, Pantoea ananatis\\u003c/em\\u003e, and \\u003cem\\u003ePantoea agglomerans\\u003c/em\\u003e. In one of the experiments conducted, we used the same set of wheat genotypes and inoculated across environments to assess the consistency of this protocol, symptoms and host response. In wheat, inoculation with \\u003cem\\u003eXtu\\u003c/em\\u003e produced characteristic bacterial leaf streak (BLS) symptoms, with water-soaked lesions that later became necrotic and streak-like, particularly under favorable environmental conditions. In the inoculated field plots, clear differences between resistant and susceptible wheat genotypes were observed (Fig. 5A). The resistant and susceptible lines grown side by side demonstrated the accuracy and consistency of the inoculation protocol. Susceptible lines exhibited rapid disease progression, leading to coalesced streaks and premature leaf senescence. In severe cases, entire leaves dried early, resulting in substantial canopy damage and potential yield reduction (Fig. 5B). Moreover, blight-like symptoms extended beyond the foliage, as black chaff appeared on wheat spikes, marked by dark, water-soaked, and necrotic lesions on glumes and awns (Fig. 5C). These symptoms were most pronounced in genotypes displaying high foliar susceptibility to \\u003cem\\u003eXtu\\u003c/em\\u003e, suggesting systemic infection and pathogen movement within the plant.\\u003c/p\\u003e\\n\\u003cp\\u003eIn contrast, inoculation with \\u003cem\\u003eX\\u003c/em\\u003e. \\u003cem\\u003eprunicola\\u003c/em\\u003e had more symptoms compared to \\u003cem\\u003eXtu\\u003c/em\\u003e and \\u003cem\\u003eP. ananatis\\u003c/em\\u003e which showed the complexity of this disease, \\u003cem\\u003eX. prunicola\\u003c/em\\u003e produced more necrotic spots than translucence streak, while \\u003cem\\u003eP. ananatis\\u003c/em\\u003e symptoms were generally less severe across replicates. While chlorosis and occasional necrotic flecks were observed on \\u003cem\\u003eP. ananatis\\u003c/em\\u003e inoculated wheat genotypes, the extent of coalescing necrotic leaves did not approach to a score of 9. Thus, \\u003cem\\u003eP. ananatis\\u003c/em\\u003e strain tested, failed to induce clear streak symptoms under field conditions, indicating either weak pathogenicity or a role more consistent with opportunistic colonization.\\u003c/p\\u003e\\n\\u003cp\\u003eCollectively, these results confirms that \\u003cem\\u003eXtu\\u003c/em\\u003e remains highly virulent for both wheat and barley in the US, capable of causing both foliar BLS and black chaff on spikes and with new species of Xanthomonas detected in the US, the BLS disease is complex and emerging in both South and North American continents (\\u003cspan class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003ch3\\u003eBlack Chaff Symptoms caused by Bacterial Leaf Streak Disease on spikes\\u003c/h3\\u003e\\n\\u003cp\\u003eIn addition to foliar BLS symptoms, clear manifestations of black chaff were observed on wheat spikes following inoculation with \\u003cem\\u003eXtu\\u003c/em\\u003e. Symptoms included dark brown to black streaks and blotches on the glumes and awns, with lesions often extending longitudinally along spikelet (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA). In severely affected spikes, symptoms intensified, with coalesced streaks and widespread necrosis on glumes and awn, giving a pronounced blackened appearance (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB).\\u003c/p\\u003e\\n\\u003cp\\u003eThe severity of black chaff was positively correlated with foliar susceptibility, as highly susceptible lines exhibiting intense BLS also showed more severe black chaff on spikes. Inoculations with \\u003cem\\u003eX. prunicola\\u003c/em\\u003e, \\u003cem\\u003ePantoea ananatis\\u003c/em\\u003e, and \\u003cem\\u003ePantoea agglomerans\\u003c/em\\u003e did not reproduce typical black chaff symptoms, producing either mild discoloration or no visible spike damage. These findings confirm that black chaff is a diagnostic symptom of \\u003cem\\u003eX\\u003c/em\\u003etu infection in wheat and highlight its importance as a phenotypic marker for assessing susceptibility and yield impact under field conditions.\\u003c/p\\u003e\\n\\u003ch3\\u003eApplication of Developed Methodology in Controlled Environment\\u003c/h3\\u003e\\n\\u003cp\\u003eThe inoculation methodology successfully induced consistent BLS symptoms under greenhouse conditions. Water-soaked lesions appeared within three days of inoculation, progressing to chlorosis and necrosis within 7\\u0026ndash;10 days. Disease severity, scored on a 1\\u0026ndash;9 scale, showed a wide range of responses across genotypes. Violin plots (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003eA, B) demonstrated clear separation between resistant, moderately resistant, and susceptible groups. Resistant genotypes were concentrated at lower scores (\\u003cspan class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e), while susceptible lines showed higher distributions (\\u003cspan class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u0026ndash;\\u003cspan class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). The statistical analysis confirmed these phenotypic differences. In Experiment 1, variation due to treatment was marginally significant (F\\u0026thinsp;=\\u0026thinsp;2.88, p\\u0026thinsp;=\\u0026thinsp;0.0571), indicating some experimental influence (\\u003cstrong\\u003eSupplementary Table\\u0026nbsp;3\\u003c/strong\\u003e). However, in Experiment 2, highly significant differences were observed among genotypes (F\\u0026thinsp;=\\u0026thinsp;7.30, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;2e\\u0026thinsp;\\u0026minus;\\u0026thinsp;16), with genotype effects explaining most of the variance compared to residual error (\\u003cstrong\\u003eSupplementary Table\\u0026nbsp;4\\u003c/strong\\u003e). These findings demonstrate that while environmental or experimental variation can influence disease expression, the methodology consistently discriminates among resistant and susceptible genotypes under greenhouse conditions.\\u003c/p\\u003e\\n\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003eField Experiments\\u003c/h2\\u003e\\n \\u003cp\\u003eField validation under natural infection pressure supported the robustness of the developed method. Both flag and bottom leaves were evaluated, with bottom leaves generally exhibiting higher severity scores, reflecting natural disease progression (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA, B). The bar chart (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eA) showed that most genotypes clustered around moderate severity classes (\\u003cspan class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e), while violin plots (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eB) revealed clear distinctions between resistant and susceptible lines.\\u003c/p\\u003e\\n \\u003cp\\u003eANOVA further confirmed significant variation among experimental factors. In the first field trial, strong effects of trial (F\\u0026thinsp;=\\u0026thinsp;67.98, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;2e\\u0026thinsp;\\u0026minus;\\u0026thinsp;16) and day (F\\u0026thinsp;=\\u0026thinsp;1296.0, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;2e\\u0026thinsp;\\u0026minus;\\u0026thinsp;16) were detected, with a smaller but significant trial \\u0026times; day interaction (F\\u0026thinsp;=\\u0026thinsp;2.74, p\\u0026thinsp;=\\u0026thinsp;0.0118) (\\u003cstrong\\u003eSupplementary Table\\u0026nbsp;5\\u003c/strong\\u003e). These results indicate that disease development was influenced by both environment and scoring time, but the magnitude of genotypic effects remained much stronger. In Experiment II, highly significant differences among wheat lines were observed (F\\u0026thinsp;=\\u0026thinsp;2.13, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;2e\\u0026thinsp;\\u0026minus;\\u0026thinsp;16), confirming that genetic variability was the primary determinant of BLS severity (\\u003cstrong\\u003eSupplementary Table\\u0026nbsp;6\\u003c/strong\\u003e).\\u003c/p\\u003e\\n \\u003cp\\u003eTogether, these results demonstrate that the methodology is reproducible in both greenhouse and field conditions, allowing reliable identification of resistant, moderately resistant, and susceptible genotypes. The combined evidence from distribution patterns and ANOVA strongly supports its use in large-scale resistance screening programs.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe lack of a reliable and reproducible inoculation protocol has long been a major constraint in advancing research on bacterial leaf streak (BLS) and black chaff of small grains. The inability to consistently reproduce infections under controlled conditions has limited progress in elucidating host-pathogen interactions, identifying resistance sources, and accelerating breeding for resistant cultivars. Although BLS has been recognized in the United States for several decades, it has emerged as a serious threat in the Northern Great Plains in recent years (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). Despite increasing importance, reproducible inoculation methods remain challenging, with most previous approaches such as needleless syringe or vacuum infiltration and leaf clipping, requiring specialized facilities and operator skill while yielding variable amounts of bacteria inoculum, disease severity outcomes, labor intensive and time-consuming (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e). The disease rating scales currently published consider only leaf water-soaking and chlorosis and do not account for necrosis symptoms. Such inconsistency in both inoculation protocols, disease severity and rating scales has hindered the fulfillment of Koch\\u0026rsquo;s postulates and slowed systematic screening for resistance. Earlier studies demonstrated that inoculation method, bacterial concentration, and plant growth stage significantly influence symptom development and disease severity. Syringe or vacuum infiltration methods force bacterial entry into leaf apoplast but often cause mechanical injury and fail to represent natural infection capabilities of bacterial strains. Conversely, spray inoculations more closely mimic field conditions but often result in uneven droplet distribution and poor bacterial adherence to the leaf surface, particularly when surfactants are absent. Environmental parameters such as humidity, temperature, and dew duration after inoculation also critically affect lesion formation [1, 3]. In several reports, bacterial suspensions below 10⁵ CFU mL⁻\\u0026sup1; failed to produce consistent symptoms, whereas higher concentrations (\\u0026ge;\\u0026thinsp;10⁶ CFU mL⁻\\u0026sup1;) induced typical streaking in susceptible cultivars (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e). These findings underscore the importance of standardizing inoculum preparation, delivery, and environmental conditions for reliable infection establishment.\\u003c/p\\u003e\\u003cp\\u003eThe air-pressure spray inoculation method described in this study addresses all of these limitations by providing a scalable and reproducible approach suitable for both controlled and field environments. Incorporating a non-ionic surfactant into the bacterial suspension ensures uniform wetting and adhesion of droplets to leaf surfaces, facilitating bacterial penetration through stomata and cuticular openings. The controlled air pressure allows consistent droplet size and uniform distribution of inoculum across leaves and spikes, producing reproducible symptom development across genotypes. In susceptible cultivars, water-soaked lesions typically appeared three days post-inoculation and progressed to chlorosis and necrosis within seven days, whereas resistant lines exhibited minute, restricted lesion expansion. This consistency supports the method\\u0026rsquo;s utility for screening germplasm and differentiating resistance levels. Notably, when applied at booting, the protocol also successfully reproduced black chaff symptoms on spikes, providing a comprehensive assessment of both foliar and reproductive stage susceptibility, a feature absent from many previous studies (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eThe broader applicability of this inoculation system was demonstrated by its successful adaptation to other bacterial pathogens, including \\u003cem\\u003eX\\u003c/em\\u003e. \\u003cem\\u003eprunicola\\u003c/em\\u003e and \\u003cem\\u003eP. ananatis.\\u003c/em\\u003e While \\u003cem\\u003eXtu\\u003c/em\\u003e remains the primary causal agent of severe BLS in U.S. wheat, emerging reports of \\u003cem\\u003eX. prunicola\\u003c/em\\u003e and \\u003cem\\u003ePantoea\\u003c/em\\u003e species highlight a complex disease etiology (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). The adaptability of the current protocol to multiple pathogens emphasizes its flexibility and potential use for comparative pathogenicity testing and cross-host inoculation studies in small grains. A further contribution of this work is the standardization of disease scoring. Previous studies often employed a 1\\u0026ndash;5 scale that captured early water soaking and chlorosis but failed to represent advanced necrotic or spike symptoms (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). The 1\\u0026ndash;9 scale implemented here provides higher resolution and aligns BLS assessment with scoring systems used for other major wheat diseases such as stripe rust, stem rust, soil-borne mosaic virus and spot blotch. This harmonization enables comparative evaluation of resistance across diseases and improves the integration of phenotypic data into practical applied pathology and breeding. Despite these advantages, the method requires careful management of inoculum quality, environmental humidity, and plant growth stage to ensure optimal results. Field disease severity was greatest when inoculation occurred at booting, suggesting that physiological and anatomical changes during spike emergence influence bacterial colonization. This observation aligns with previous reports that host developmental stage affects infection success and symptom expression (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). Further refinement is needed to define the latent epiphytotic period, quantify bacterial proliferation within tissues during pathogenic stage, and assess the influence of environmental variability across locations.\\u003c/p\\u003e\\u003cp\\u003eOverall, the air-pressure spray inoculation method represents a significant methodological advance for BLS and black chaff research. Its reproducibility, scalability, and adaptability make it suitable for both fundamental studies on \\u003cem\\u003eXanthomonas translucens\\u003c/em\\u003e pathogenesis and large-scale resistance screening in pathology and breeding research. Moreover, the approach can be extended to related cereal hosts and emerging bacterial pathogens, positioning it as a versatile and standardized tool for the wider plant research community.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study establishes a reliable, reproducible, and cost-effective assay for bacterial leaf streak (BLS) evaluation in wheat. The protocol consistently reproduced characteristic symptoms under both greenhouse and field conditions, enabling clear differentiation between resistant and susceptible genotypes using a standardized 1 to 9 scale. Multiple repetitions confirmed the robustness, accuracy and adaptability of this method, not only for \\u003cem\\u003eXtu\\u003c/em\\u003e but also for related pathogens such as \\u003cem\\u003eX. prunicola\\u003c/em\\u003e and \\u003cem\\u003eP. ananatis\\u003c/em\\u003e. Unlike earlier approaches that were not accurate, labor-intensive, or dependent on specialized equipment, this assay is accurate, simple, scalable, and efficient for large germplasm screening. By integrating greenhouse and field evaluations, the protocol provides a powerful tool to accelerate discovering key components of the pathogen effectors, host resistance genes, plant-bacterial interactions, yield loss estimation, and breeding efforts aimed at developing BLS-resistant varieties. Importantly, it also demonstrates potential utility for other cereals and bacterial diseases, broadening its impact on cereal pathology and crop improvement.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eKey Implications\\u003c/h2\\u003e\\u003cp\\u003eThe development of this simple yet robust assay directly benefits plant biologists, breeders, pathologists, and agronomists by offering an efficient way to screen wheat germplasm for resistance against BLS. Its flexibility across pathogens and cereal crops provides a unified framework for studying streak-like bacterial diseases, which are increasingly threatening cereal production worldwide. Most importantly, this protocol bridges-controlled environment and field evaluations, ensuring reliable disease phenotyping that can accelerate research. Ultimately, the adoption of this method will contribute to accelerated breeding for BLS resistance, sustainable wheat production, reduced yield losses, and enhanced global food security.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Material and Methods\",\"content\":\"\\u003cp\\u003e\\u003cb\\u003eXtu\\u003c/b\\u003e \\u003cb\\u003eIsolation and Characterization\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe diseased samples were collected from the Volga SDSU wheat field station and the Dakota Lakes Research Farm on July 9th, 2023. The diseased samples were cut into small pieces and surface sterilized with 10% bleach and 70% ethanol, followed by ddH2O washing (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e). The leaf was crushed into ddH\\u003csub\\u003e2\\u003c/sub\\u003eO and with the help of a sterile loop, streaked on nutrient agar (NA) (\\u003cb\\u003eSupplementary Table\\u0026nbsp;1\\u003c/b\\u003e) and kept in an incubator for 2 days at 28\\u0026deg;C. The single yellowish colony streaked separately on NA plates for pure culture. The DNAs were extracted from all single colonies by using the protocol of (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e) for Multiplex primer amplification specific to \\u003cem\\u003eXanthomonas translucence\\u003c/em\\u003e spp. and 16S rRNA gene sequencing.\\u003c/p\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCulture Preparation and Greenhouse Inoculation\\u003c/h2\\u003e\\u003cp\\u003eAfter the confirmation of \\u003cem\\u003eXtu AL-1029\\u003c/em\\u003e (Accession No. PQ524066) (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e) the secondary culture was grown in NA media supplemented with 10% sucrose. Bacterial cells were then suspended in 1X phosphate saline buffer (PBS) (pH 7.4) to prepare an inoculum. To achieve an optical density (OD\\u003csub\\u003e600\\u003c/sub\\u003e) of 0.4, or around 1\\u0026times;10\\u003csup\\u003e8\\u003c/sup\\u003e cfu/ml, the cell concentration was adjusted. Four drops of Tween-20 (polyoxyethylene sorbitan monolaurate, Sigma-Aldrich) were added as a surfactant to the bacterial solution before inoculation. The plants were grown in 9-inch cones with three replications, and each replication had two plants, total six plants were inoculated after 15 days of germination. The bacterial solution was equally sprayed over the plants till runoff for the real inoculation procedure. A spray cannon connected to an air pump with around 20psi of pressure. Following this, the infected plants were kept at room temperature and subjected to a 12-hour photo period in misting chambers for two days. The plants were moved and placed on flow trays within the greenhouse room at 80% humidity for seven days. The assessment of disease development was scored by a scale from 1 to 9 (1 for highly Resistant and 9 for highly susceptible), with an estimation made of the proportion of the plant area affected by the disease.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eField Inoculation Assay\\u003c/h2\\u003e\\u003cp\\u003eThe same protocol was used for inoculating the field trial. Inoculation was carried out at the wheat booting stage using an air-pressure sprayer, without the addition of carborundum powder. Disease severity was assessed at 7th, 14th, and 21st days post-inoculation (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;1\\u003c/b\\u003e), scoring the flag leaf on a 1\\u0026ndash;9 BLS disease scale. The same method was applied to screen \\u003cem\\u003eXanthomonas prunicola\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e) and \\u003cem\\u003ePantoea ananatis\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e) in the field. Since BLS is a disease complex, this protocol can be reliably used to evaluate all bacterial pathogens that produce streak-like symptoms on small grains plants. In summer 2024 and 2025, more than 1000 row plots of winter, spring and durum wheat and winter barley were inoculated with \\u003cem\\u003eAL-1029 Xtu\\u003c/em\\u003e strain by following the same protocol. There was one repeated experiment in 2024 and 2025 where the same set of genotypes was inoculated with \\u003cem\\u003eXtu\\u003c/em\\u003e, \\u003cem\\u003eP. ananatis\\u003c/em\\u003e and \\u003cem\\u003eX. prunicola\\u003c/em\\u003e by following the same protocol.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eDevelopment of the new Methodology\\u003c/h2\\u003e\\u003cp\\u003eHere, we provide a detailed, step-by-step procedure for small-scale, repeatable BLS infection testing in wheat. The procedure was created to treat wheat that had been infected with \\u003cem\\u003eXanthomonas translucence\\u003c/em\\u003e spp. It comprises a pre-propagation and simple harvesting process to generate a large enough quantity for an infection trial. The technique may be quickly established because of the thorough process of knowledge and ease of handling. The necessary tools are easily accessible and reasonably priced. In addition, we show that a particular resistance phenotype may be evaluated by combining our infection strategy with macroscopic and microscopic assessment techniques. Finally, the procedure is easily adaptable to various host plants and bacterial and fungal diseases.\\u003c/p\\u003e\\u003cp\\u003e\\u003cul\\u003e\\u003cli\\u003e\\u003cp\\u003eDiseased wheat leaves showing typical streak and necrosis symptoms were collected from the field (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eA).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eCollected leaves were cut into small pieces, surface-sterilized to eliminate epiphytic microbes, and macerated in 500 \\u0026micro;L sterile water. The suspension was streaked onto nutrient agar (NA) plates and incubated at 28\\u0026deg;C for 2 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eB).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eSingle colonies were sub-streaked onto fresh NA plates to establish pure cultures; a process repeated three times to ensure purity. The identity of \\u003cem\\u003eX. translucens pv. undulosa\\u003c/em\\u003e (\\u003cem\\u003eXtu\\u003c/em\\u003e) was confirmed to be using published multiplex primers (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e) and agarose gel electrophoresis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eC).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003ePure \\u003cem\\u003eXtu\\u003c/em\\u003e cultures were streaked onto NA plates supplemented with 10% sucrose and incubated for 5\\u0026ndash;7 days to obtain maximum bacterial growth prior to inoculation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eD).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eA 1\\u0026times; phosphate-buffer saline (PBS) solution (\\u003cb\\u003eSupplementary Table\\u0026nbsp;2\\u003c/b\\u003e) was prepared and autoclaved. Bacterial colonies were scraped from the NA plates, resuspended in 1X PBS, and adjusted to an optical density (OD\\u003csub\\u003e600\\u003c/sub\\u003e) of ~\\u0026thinsp;0.4, corresponding to 1 \\u0026times; 10\\u003csup\\u003e8\\u003c/sup\\u003e cfu/mL. Four drops of Tween-20 were added per 100 mL of inoculum (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eE).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eWheat plants were grown in 9-inch cones, each containing two seeds, with three replications (total: 6 plants per treatment). Plants were maintained under controlled conditions (23\\u0026ndash;25\\u0026deg;C, 14 h light/10 h dark photoperiod) for 15 days, with adequate watering and fertilizer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eF).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eInoculation was performed 15 days after germination. The prepared inoculum was applied evenly to the plants using an air sprayer set at 25 psi (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eG).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eInoculated plants were incubated in a humidity chamber at 25\\u0026deg;C with 100% relative humidity and a 14 h light/10 h dark cycle for 48 h (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eH).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eFollowing incubation, plants were transferred to a transparent tent maintained at ~\\u0026thinsp;80% relative humidity with the aid of a humidifier, where they remained for 7\\u0026ndash;10 days to allow disease development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eI).\\u003c/p\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cp\\u003eDisease severity was assessed 7 days post-inoculation using a 1\\u0026ndash;9 rating scale (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) initial symptoms were gummy like exudates produced on leaf surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eJ, K), these exudates progressed into translucence streak (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eL) which later turned necrotic lesion (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eM).\\u003c/p\\u003e\\u003c/li\\u003e\\u003c/ul\\u003e\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003eBLS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003eBacterial leaf streak\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eXtu\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eXanthomonas\\u003c/em\\u003e \\u003cem\\u003etranslucence\\u003c/em\\u003e pathovar \\u003cem\\u003eundulosa\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eXp\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eXanthomonas prunicola\\u003c/em\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003ePBS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003ePhosphate buffer saline\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003ecfu\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003eColony forming unites\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003eAL1029\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003eAmeen Lab strain 1029\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003eDPI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003eDay post Inoculation\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003eNA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003eNutrient agar\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 16.8874%;\\\"\\u003e\\n \\u003cp\\u003eSDSU\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 83.1126%;\\\"\\u003e\\n \\u003cp\\u003eSouth Dakota State University\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements:\\u0026nbsp;\\u003c/strong\\u003eWe thank Mr. Jack Ingemansen, Steve Stein, Cody Hall, Chris Nelson, Julie Thomas and Dr. Dalitso Yabwalo for help with field planting, preparations and management of research plots.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors Information:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAuthors and Affiliations\\u003c/p\\u003e\\n\\u003cp\\u003eDepartment of Agronomy, Horticulture \\u0026amp; Plant Science, South Dakota State University, Brookings, South Dakota, 57007, U.S.A.\\u003c/p\\u003e\\n\\u003cp\\u003eMuhammad Ahmad, Tapish Pawar, Shyam Solanki, Karl Glover and Gazala Ameen\\u003c/p\\u003e\\n\\u003cp\\u003eContributions\\u003c/p\\u003e\\n\\u003cp\\u003eMA planned and designed the experiments, conducted research, analyzed the data, prepared figures and wrote the manuscript. GA, MA and KG develop the protocol. MA and TP conducted the field trials and collected data. GA and KG gathered funding for the projects. SS, TP, KG and GA reviewed the manuscript, edited and provided the ideas for improvement of the technique and manuscript. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCorresponding author\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCorrespondence to Gazala Ameen\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was carried out with the support of the Agricultural Experimentation Funding SD00H754, SD00H717, and South Dakota Wheat Commission funding SA2400069 and SA2500056.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics Declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEthics approval and consent to participate\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003eConsent for Publication\\u003c/p\\u003e\\n\\u003cp\\u003eAll the authors have reviewed and edited this manuscript and agreed to submit it to the Plant Method Journal for publication. This manuscript have not been submitted to any other journals and has not published in any other journal.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that there are no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eSapkota S, Harris-Shultz KR, Strickland TC, Anderson WF. Identification of cultured and diazotrophic bacterial endophytes in warm-season grasses. PhytoFrontiers\\u0026trade;. 2023;3(2):411\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eRom\\u0026aacute;n-Reyna V, Curland RD, Velez-Negron Y, Ledman KE, Gutierrez Castillo DEE, Beutler J et al. Development of genome-driven, lifestyle-informed markers for identification of the cereal-infecting pathogens Xanthomonas translucens pathovars undulosa and translucens. Phytopathology. 2022(ja).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eKandel YR, Glover KD, Tande CA, Osborne LE. Evaluation of spring wheat germplasm for resistance to bacterial leaf streak caused by Xanthomonas campestris pv. translucens. Plant Dis. 2012;96(12):1743\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eRamakrishnan SM, Sidhu JS, Ali S, Kaur N, Wu J, Sehgal SK. Molecular characterization of bacterial leaf streak resistance in hard winter wheat. PeerJ. 2019;7:e7276.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFriskop A, Green A, Ransom J, Liu Z, Knodel J, Hansen B, et al. Increase of bacterial leaf streak in hard red spring wheat in North Dakota and yield loss considerations. Phytopathology\\u0026reg;. 2023;113(11):2103\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSilva IT, Rodrigues F\\u0026Aacute;, Oliveira JR, Pereira SC, Andrade CCL, Silveira PR, et al. Wheat resistance to bacterial leaf streak mediated by silicon. J Phytopathol. 2010;158(4):253\\u0026ndash;62.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eDuveiller E, Bragard C, Maraite H. Bacterial leaf streak and black chaff caused by Xanthomonas translucens. International Maize and Wheat Improvement Center, Estado de M\\u0026eacute;xico, Mexico; 1997. pp. 25\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eDusek GD. Management Strategies for Bacterial Leaf Streak on Hard Red Spring Wheat. North Dakota State University; 2025.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHagborg W. Black chaff, a composite disease. Can J Res. 1936;14(9):347\\u0026ndash;59.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eDuveiller E. Bacterial leaf streak or black chaff of cereals. EppO Bull. 1994;24(1):135\\u0026ndash;57.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBoosalis M. A partial-vacuum technique for inoculating seedlings with bacteria and fungi. Phytopathology. 1950;40(1).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHagborg W. A device for injecting solutions and suspensions into thin leaves of plants. Can J Bot. 1970;48(6):1135\\u0026ndash;6.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAcharya K, Liu Z, Schachterle J, Kumari P, Manan F, Xu SS, et al. Genetic mapping of QTLs for resistance to bacterial leaf streak in hexaploid wheat. Theor Appl Genet. 2024;137(12):265.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eClavijo F, Curland RD, Croce V, Lapaz MI, Dill-Macky R, Pereyra S, et al. Genetic and Phenotypic Characterization of Xanthomonas Species Pathogenic in Wheat in Uruguay. Phytopathology\\u0026reg;. 2022;112(3):511\\u0026ndash;20.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAhmad M, Gonzalez Hernandez JL, Solanki S, Glover KD, Ameen G. First Report of Xanthomonas prunicola causing Leaf Streak Disease on Wheat (Triticum aestivum) in the United States. Plant Disease. 2025(ja).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLedman KE, Osdaghi E, Curland RD, Liu Z, Dill-Macky R, Epidemiology. Host Resistance, and Genomics of the Small Grain Cereals Pathogen Xanthomonas translucens: New Advances and Future Prospects. Phytopathology\\u0026reg;. 2023;113(11):2037\\u0026ndash;47.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAdhikari TB, Gurung S, Hansen JM, Bonman JM. Pathogenic and Genetic Diversity of Xanthomonas translucens pv. undulosa North Dak Phytopathology\\u0026reg;. 2012;102(4):390\\u0026ndash;402.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGomes LH, Duarte KMR, Andrino FG, Tavares FCA. A simple method for DNA isolation from Xanthomonas spp. Scientia Agricola. 2000;57:553\\u0026ndash;5.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAhmad M, Alhusays A, Galvin C, Torres M, Fitzpatrick E, Glover K, et al. editors. Bacterial leaf streak disease resistance loci identification in winter wheat. PHYTOPATHOLOGY; 2024: AMER PHYTOPATHOLOGICAL SOC 3340 PILOT KNOB ROAD, ST PAUL, MN 55121 USA.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlhusays A, Galvin C, Torres M, Senger M, Fitzpatrick E, Gonzalez-Hernandez JL, et al. First Report of Pantoea ananatis causing leaf streak disease on wheat (Triticum aestivum) in the United States of America. Plant Dis. 2024;108(9):2913.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Bacterial Leaf Streak Disease, Wheat, Xanthomonas translucence spp. Infection Assay, Greenhouse\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8098615/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8098615/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWheat (\\u003cem\\u003eTriticum aestivum\\u003c/em\\u003e) is one of the most economically important crops in the United States. However, over the past two years, wheat production has suffered up to a 40% reduction in final yield due to pathogen infections worldwide. A major emerging threat in the Great Plains and Canadian Prairies, including South Dakota, North Dakota, and Minnesota, is bacterial leaf streak (BLS)/black chaff disease caused by \\u003cem\\u003eXanthomonas translucens\\u003c/em\\u003e spp., which has led to substantial yield losses in the last decade. Absence of both effective chemical controls and competitive highly resistant varieties makes BLS disease management very difficult. A critical step missing in this process is the establishment of a reliable and reproducible infection protocol for resistance evaluation under both controlled and field conditions. Currently, no protocols are published, and the methods published as part of research manuscripts lack detailed procedures, equipment specifications, and have major drawbacks for applications limited to controlled environment and discrepancies in field disease ratings scales. Therefore, we are presenting here a robust and reproducible BLS disease infection protocol and, disease severity rating scale for estimation of BLS disease in both controlled and field conditions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter Three days of inoculation with \\u003cem\\u003eX. translucens\\u003c/em\\u003e pv. \\u003cem\\u003eundulosa\\u003c/em\\u003e (\\u003cem\\u003eXtu\\u003c/em\\u003e), wheat plants developed initial water-soaked symptoms at inoculation sites. Over seven days, symptoms progressed to chlorosis and necrosis, frequently covering entire leaves of highly susceptible genotypes, whereas limited to no symptoms on resistant genotypes. Disease severity was consistently scored on a 1–9 scale, enabling clear differentiation of resistant, moderately resistant, and susceptible genotypes. Pathogen re-isolation confirmed infection fidelity. Field validation at the booting stage produced comparable symptom progression on flag leaves, with severity scored at 7, 14, and 21 days post-inoculation. The same protocol was successfully adapted for \\u003cem\\u003ePantoea ananatis\\u003c/em\\u003e and \\u003cem\\u003eXanthomonas prunicola\\u003c/em\\u003e, demonstrating the adaptability of the method. The protocol was repeated across five independent trials and produced reproducible results in both controlled and field environments.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe describe a simple, reproducible, and cost-effective inoculation protocol for evaluating BLS severity in wheat. The method reliably distinguishes resistance responses across environments and can be extended to other bacterial pathogens affecting small grains. Its affordability, accessibility, and reproducibility make it a valuable tool for large-scale germplasm screening and resistance breeding.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eKey Features\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e• A detailed and systemic infection protocol is devised for different cultivars of wheat.\\u003c/p\\u003e\\n\\u003cp\\u003e• Plants can screen at seedling and adult-plant stage.\\u003c/p\\u003e\\n\\u003cp\\u003e• No specific equipment required.\\u003c/p\\u003e\\n\\u003cp\\u003e• Using an inexpensive pipeline to ensure uniform symptoms.\\u003c/p\\u003e\\n\\u003cp\\u003e• This protocol is validated for other bacterial species that are reported to cause bacterial leaf streak symptoms on small grains (\\u003cem\\u003ePantoea\\u003c/em\\u003e spp and \\u003cem\\u003eXanthomonas prunicola\\u003c/em\\u003e on small grains (wheat and Barley).\\u003c/p\\u003e\",\"manuscriptTitle\":\"An Effective Method for Bacterial Leaf Streak Disease Severity Estimation in Controlled and Field Environments in Small Grains\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-24 18:26:26\",\"doi\":\"10.21203/rs.3.rs-8098615/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"02765418-2650-4ea1-a372-034fe898d99a\",\"owner\":[],\"postedDate\":\"November 24th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-12-10T22:53:14+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-11-24 18:26:26\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8098615\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8098615\",\"identity\":\"rs-8098615\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}